This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Introduction

So far, no effective therapy is available for acute kidney injury (AKI), a common
and serious complication with high morbidity and mortality. Interest has recently
been focused on the potential therapeutic effect of mouse adult renal progenitor cells
(MRPC), erythropoietin (EPO) and suramin in the recovery of ischemia-induced AKI.
The aim of the present study is to compare MRPC with MRPC/EPO or MRPC/suramin concomitantly
in the treatment of a mouse model of ischemia/reperfusion (I/R) AKI.

Results

MRPC exhibited characteristics consistent with renal stem cells. The features of MRPC
were manifested by Pax-2, Oct-4, vimentin, α-smooth muscle actin positive, and E-cadherin
negative, distinguished from mesenchymal stem cells (MSC) by expression of CD34 and
Sca-1. The plasticity of MRPC was shown by the ability to differentiate into osteoblasts
and lipocytes in vitro. Injection of MRPC, especially MRPC/EPO and MRPC/suramin in I/R AKI mice attenuated
renal damage with a decrease of the necrotic injury, peak plasma Cr and BUN. Furthermore,
seven days after the injury, MRPC/EPO or MRPC/suramin formed more CD34+ and E-cadherin+ cells than MRPC alone.

Conclusions

These results suggest that MRPC, in particular MRPC/EPO or MRPC/suramin, promote renal
repair after injury and may be a promising therapeutic strategy.

Keywords:

Adult kidney stem cell; Cell therapy; Erythropoietin; Suramin

Introduction

Acute kidney injury (AKI) mainly develops following ischemic or toxic insults and
is characterized by acute tubular injury and renal dysfunction [1,2]. Modern dialysis techniques, such as intermittent or continuous renal replacement
therapy, are used in the treatment of AKI, but the syndrome is still characterized
by a high mortality and morbidity rate [3]. Thus, it is urgent for us to identify new drugs and find novel therapeutic strategies.

Recently, stem cell therapy has been proposed as a promising alternative in the treatment
of AKI [4-8], due to the highly versatile response of cells to their environment. The potential
use of stem cells in regenerative medicine to treat kidney diseases represents a critical
clinical goal [9]. Mounting evidence indicates that stem cells from different sources have therapeutic
potential for AKI, including bone marrow-derived stem cells [4-7], embryonic stem cells, induced pluripotent stem cells, human amniotic fluid stem
cells [10], human cord-blood stem cells [11] and resident renal stem cells [8]. Among these stem cells, little is known about renal stem cells in the treatment
of AKI, because their localization, markers, function and mechanism are still not
fully understood. Recent study focuses on an important role of renal stem cells in
the treatment of AKI by the mechanism of differentiating into renal tubule cells [12-14]. Especially, mouse renal stem cells accelerate renal regeneration and prolong survival
after AKI by differentiating into renal tubule cells and vessel endothelial cells
with the expression of E-cadherin and CD34 [15]. This potentially gives a clue to the development of regenerative medicine in the
treatment of human renal diseases. Although many efforts have been made to investigate
renal stem cells in the treatment of AKI, therapy with renal stem cells for AKI treatment
needs more research.

Besides stem cell-based therapy, drug therapy is also applied in the recovery of renal
ischemia/reperfusion (I/R) injury. Thus, exploring new drugs or novel pharmacological
effects of known drugs in the treatment of AKI is urgent. Recently, erythropoietin
(EPO) and suramin were intensely studied in the treatment of AKI for their novel pharmacological
effect. EPO may have tissue-protective properties in addition to its well-known erythropoietic
function [16]. Song YR et al. [17] report that preventive administration of EPO could prevent AKI and improve postoperative
renal function. EPO may preserve kidney integrity and reinforce the regeneration of
tubular epithelium by anti-apoptotic and anti-inflammatory features [18]. Suramin, a polysulfonated naphthylurea usually given in humans in the treatment
of trypanosomiasis, is reported to accelerate recovery from renal dysfunction caused
by IR injury in mice [19,20]. The mechanisms remain incompletely understood and may be related to prevention of
apoptosis, inhibition of inflammatory cytokine generation and facilitating epithelial
cell hyperplasia [19].

In this study, we explored the different effects of mouse adult renal progenital cells
(MRPC) alone or MRPC/EPO or MRPC/suramin in the treatment of AKI. Mouse renal MRPC
which were isolated from adult GFP mice, possessed features consistent with renal
stem cells. Injection of these MRPC, MRPC/EPO, or MRPC/suramin could rescue renal
damage in I/R AKI C57BL/6 mice, followed by formation of CD34+ and E-cadherin+ cells. More pronounced protection of renal function was found in mice treated with
MRPC/EPO or MRPC/suramin. Thus, MRPC, particularly MRPC/EPO or MRPC/suramin, might
be a promising therapeutic target for the treatment of AKI.

Methods

Experiments were carried out on 72 male C57BL/6 mice, with weights ranging from 27
to 32 g at the time of ischemia. C57BL/6-gfp mice were bought from the experimental
animal center of the Fourth Military Medical University. All animal procedures were
approved by the animal ethics committee of Shandong University (Jinan, China) and
followed the Guide for the Care and Use of Laboratory Animals published by the U.S.
National Institutes of Health (NIH Publication No. 85–23, revised 1996).

Cell isolation and culture

MRPC were isolated from the renal cortex of eight-week-old C57BL/6-gfp transgenic
mice (Fourth Military Medical University, Xian, China) using a previously reported
approach [8,21]. Briefly, the kidney was perfused in vivo with PBS to wash out blood and was then dissected. The renal capsule and medulla
tissue were removed and digested with 0.125% type_IV collagenase (Sigma-Aldrich, St
Louis, MO, USA) and 0.25% trypsin at 37°C for 30 minutes with gentle shaking. After
resuspension in (D)MEM/F12 Sigma-Aldrich), the fraction was filtered through a 200
μm mesh (BD Biosciences, San Jose, CA, USA) to remove undigested tissue, and then
a 40 μm mesh was used to remove smaller renal tubules and cell aggregates. The filtered
fraction was washed with (D)MEM/F12 containing 10% fetal bovine serum (FBS) (Sigma-Aldrich).
To exclude autofluorescence of isolated cells, the level of autofluorescence was detected
in similar cell preparations from C57BL/6 mice under a fluorescence microscope. To
avoid cell–cell contact, GFP-positive cells were plated at low density (300 cells/cm2) on fibronectin coated culture flasks in the (D)MEM/F12 culture medium containing
10% FBS, 100 U/ml of penicillin, 100 μg/ml of streptomycin, and incubated at 37°C
in the presence of 5% CO2.

Characterization of MRPC

Immunocytochemistry of MRPC

Cells growing on a poly-l-lysine coated 24-well plate were washed three times with
PBS and fixed in 4% paraformaldehyde for 30 minutes. Cells were permeabilized with
0.1% Triton X-100 PBS for 20 minutes and then blocked with 4% goat serum for one hour.
Then cells were incubated with primary antibodies for one hour at room temperature
in the absence of sunlight. The following primary antibodies were used: mouse monoclonal
anti-Oct-4 (Chemicon, Billerica, MA, USA, MAB4419, 1:200), rabbit polyclonal anti-Pax2
(Covance, Emeryville, CA, USA, PRB-276P, 1:400), rat monoclonal anti-E-cadherin (Chemicon,
MABT26, 1:200), mouse polyclonal anti-vimentin (Chemicon, MAB3400, 1:200) and mouse
monoclonal anti-alpha smooth muscle actin (α-SMA) antibody (Sigma-Aldrich, A2547,
1:600). After three washes with tris-buffered saline (TBS), cells were incubated with
alexa 594-conjugated secondary antibodies (Zhongshan Goldenbridge, China) in PBS.
4,6-Diamino-2-phenyl indole (DAPI) was used for nuclear counterstaining. After washing,
slides were mounted with a cover slip in Glycergel Antifade Medium (Dako, Carpinteria,
CA, USA). Negative controls were performed using non-immune IgG instead of the primary
antibodies. Images were obtained using an Olympus fluorescence microscope. Two independent
investigators evaluated the number of Oct-4-, Pax-2-, vimentin- and α-SMA-positive
MRPC by counting three randomly selected high-power fields.

Effect of MRPC on renal protection after acute ischemic injury

Study design

Twenty-four mice were randomly divided into controls (positive control) or either
of the three treatment arms (MRPC, MRPC/EPO or MRPC/suramin). Animals were housed
at a constant temperature and humidity, with a 12:12-hour light–dark cycle. At days
0 (pre-ischemia), 1, 2 and 3, blood samples were collected for the measurement of
serum creatinine (Cr) and blood urea nitrogen (BUN). Cr and BUN concentrations were
detected by the Jaffe method (Beckman Coulter Synchron LX System; Beckman Coulter
Inc., Brea, Calif., USA). Then, the mice were sacrificed at day 7. An additional 48
mice were used to observe the early changes in the kidney after injury; 24 mice (n
= 6 in each group) were sacrificed at day 2, and the other 24 mice (n = 6 in each
group) were sacrificed at day 4. Bilateral kidneys were obtained and fixed with formalin
followed by paraffin embedding. Sections were stained with H & E and studied histologically
for morphologic changes induced by ischemic injury. A grading scale (range: 0 to 4)
for assessment of acute tubular necrosis developed by Jablonski et al. was used for the histopathological assessment of acute ischemic injury [23]. In addition, immunohistochemistry assays were performed with anti-GFP antibodies
to detect and localize the infused stem cells in the tissue as well as the expression
level of E-cadherin and CD34 after treatment.

Surgical procedure

Mice were anesthetized with an intraperitoneal injection of phenobarbital (150 μg/g).
An abdominal midline incision was made to expose the kidneys and nontraumatic vascular
clamps were used to clamp both renal pedicles for 30 minutes at room temperature.
After visual reflow of both kidneys, 50 μl of cell suspensions containing 5 × 105 MRPC in PBS or MRPC/EPO (administration of 5 × 105 MRPC in 50 μl PBS and 1 μg/kg of EPO) or MSC/suramin (administration of 5 × 105 MRPC in 50 μl PBS and 1 mg/kg of suramin) were injected immediately and slowly through
the tail vein after surgery. Mice in the control group received 50 μl of PBS only.

Statistical analysis

Data are shown as means ± SD. Comparison between groups was evaluated by two-way analysis
of variance (ANOVA) or unpaired t test. P <0.05 was considered statistically significant.

Results

Isolation and culture of fluorescent MRPC

MRPC were isolated from six- to eight-week old C57BL/6-gfp mice. Cells from six- to
eight- week old C57BL/6 mice were used as controls for autofluorescence detection.
Autofluorescence was negligible in cells from C57BL/6 mice as detected by fluorescence
microscopy (Figure 1A). Dispersed cells from C57BL/6-gfp mice became monomorphic and had a spindle-shaped
appearance after four weeks of culture (Figure 1A). These cells containing a large nucleus and scant cytoplasm, expressed green fluorescence
at different passages (Figure 1A). After more than 50 passages, there was no evidence of senescence in some clones.
MRPC between 15 and 20 passages were used in the study.

Figure 1.Characteristics and differentiation potency of MRPC. (A) Morphology of MRPC isolated from adult mouse kidney. Morphology of the cells after
1 passage (4 days), 8 passages, 25 passages, and 50 passages is shown by phase-contrast
microscopy and immunofluorescence microscopy. After eight passages, the cells are
monomorphic with a spindle-shape morphology, containing large nucleus and fluorescence.
MRPC cultured to confluence at passage 25 do not overlay and maintain fluorescence.
Autofluorescence was negligible in the control group. (magnification 200×) (B) Immunofluorescence microscopy of fluorescent MRPC (green) stained with the following
antibodies (red): anti-Oct-4 antibodies, anti-Pax-2 antibodies, an anti-alpha smooth
muscle actin antibody (α-SMA), an anti-vimentin antibody, an anti-E-cadherin antibody
and secondary antibody only (magnification 400×). (C) Gene expression of mMSC (bone marrow) and MRPC detected by RT-PCR. (D) Mutilineage differentiation of MRPC. Phase-contrast microscopy and immunofluorescence
microscopy of MRPC that were incubated under culture conditions that induced differentiation
into osteoblasts and adipocytes. Control A was osteogenic differentiation and control
B was adipogenic differentiation (magnification 100×). MRPC, mouse renal progenitor
cells; MSC, mesenchymal stem cells, α-SMA.

Expression of renal progenitor cell markers in MRPC

MRPC expressed Oct-4, Pax-2, α-SMA and vimentin but not E-cadherin as shown by the
immunocytochemistry assay (Figure 1B). Furthermore, MSC from the bone marrow of C57BL/6 mice (mMSC) were isolated to
identify the different phenotypes between mMSC with MRPC. Many markers of renal progenitors
were expressed in MRPC but not mMSC as assessed by RT-PCR, including Oct-4, Pax-2,
Wnt-4 and WT-1. However, CD-34 and Sca-1 were expressed in mMSC but not MRPC (Figure 1C). These results indicated that MRPC are kidney progenitor cells.

Differentiation potential of MRPC

The in vitro differentiation capacity of MRPC was examined to investigate further the potency
of MRPC. When induced by osteogenic differentiation medium, MRPC stained positive
with Alizarin Red, indicating that they underwent osteogenic differentiation in vitro (Figure 1D). MRPC treated with adipogenic differentiation medium showed the presence of adipocyte
morphology with positive staining for Oil-Red O (Figure 1D), which indicated their ability for adipocyte differentiation. Taken together, multi-differentiation
function in vitro showed that MRPC were pluripotent.

To investigate whether MRPC, MRPC/EPO or MRPC/suramin have beneficial effects on regeneration
after AKI, renal histology and function were studied in I/R AKI C57BL/6 mice that
had received tail-vein injections of MRPC, MRPC/EPO, MRPC/suramin or PBS immediately
after the reperfusion. MRPC-, MRPC/EPO- and MRPC/suramin-treated mice (treatment groups)
showed a reduction in the infarct zone of the injured kidney in comparison with the
PBS- treated mice (positive control) (Figure 2A). Moreover, a better preservation of renal structure was shown in MRPC-, MRPC/EPO-
and MRPC/suramin-treated mice (Figure 2B-N). Kidneys of the positive controls exhibited severe capillary congestion and necrosis
of the tubular epithelium (Figure 2C) at day 2 and marked tubular edema and obstruction with cellular debris (Figure 2G) at day 4; and some regenerating renal tubular cells with vacuoles still appeared
in the tubular injury at day 7 (Figure 2K). However, decreased histological features of necrotic injury after ischemia were
sharply revealed in the kidneys of the treatment groups (Figure 2H-J). More regenerating renal tubular cells with brush border repaired tubular injury
was followed by the disappearance of most necrotic tubules at day 7 (Figure 2L-N), especially in MRPC/EPO- and MRPC/suramin-treated mice. Quantitative analysis
of renal tubular necrosis using the grading scores of Jablonski et al. [23] is shown in Figure 2O. Severe acute tubular necrosis in the kidneys of positive controls, compared to
the treatment groups (especially MRPC/EPO- and MRPC/suramin-treated mice) was shown
by histological grading at two days after renal ischemia (grading scores, MRPC versus
positive control, MRPC/EPO versus positive control, MRPC/suramin versus positive control,
P <0.01; MRPC/EPO versus MRPC, P <0.05; MRPC/suramin versus MRPC, P <0.01; MRPC/EPO versus MRPC/suramin, P >0.05).

Besides a better preservation of renal structure, improvement of renal function was
observed in MRPC-, especially MRPC/EPO- and MRPC/suramin-treated mice. Serum Cr and
BUN levels were measured in the treatment groups and positive controls at day 0, 1,
2 and 3. Cr and BUN reached their peak levels at day 2 of renal I/R injury in all
groups. However, significantly lower levels of Cr were detected in treatment groups,
especially MRPC/EPO- and MRPC/suramin-treated mice, compared to that of the positive
control at day 1, 2 and 3 (Figure 2P). Taken together, MRPC alone, MRPC/EPO and MRPC/suramin were more effective in improving
kidney structure and function of I/R AKI mice; MRPC/EPO and MRPC/suramin had more
therapeutic effects than MRPC alone.

Localization and roles of MRPC, MRPC/EPO and MRPC/suramin in mice with AKI

It is reported that mouse kidney progenitor cells (MKPC) accelerate renal regeneration
and prolong survival after ischemic injury by differentiation mechanisms in which
some MKPC formed vessels with red blood cells inside (CD34+ cells) and some incorporated into renal tubules (E-cadherin+ cells) [15]. To further study the localization and roles of MRPC, MRPC/EPO and MRPC/suramin in
the treatment of AKI, immunochemistry staining was performed to trace MRPC by staining
GFP and analyzing the roles of MRPC, MRPC/EPO and MRPC/suramin after injection in
I/R AKI C57BL/6 mice at day 2, 4 and 7 after ischemic injury (Figures 3, 4 and 5). GFP+ cells can become lodged in the interstitium of the kidney on day 2, 4 and 7. As shown
in Figures 3, 4 and 5, CD34+ and E-cadherin+ cells were formed when MRPC, MRPC/EPO or MRPC/suramin were injected after ischemic
injury. There were abundant E-cadherin and CD34 positive cells formed in the interstitium
of kidney at day 2 (Figure 3). Wider distribution of E-cadherin and CD34 positive cells was shown in MRPC/EPO-
and MRPC/suramin- than MRPC- treated groups at day 4 (Figure 4). The positive area decreased in the MRPC/EPO and MRPC/suramin groups, while it still
remained wide in the MRPC group at day 7 (Figure 5). These results revealed that MRPC/EPO and MRPC/suramin promoted renal function recovery
very early (day 2) after injection with their fast incorporation into renal tubules
and capillaries; however, MRPC alone played a sustaining renal repair role in I/R
AKI C57BL/6 mice.

Figure 3.Expression of GFP, CD34, and E-cadherin in consecutive sections of the kidney at day
2 after I/R injury. GFP, E-cadherin and CD34 positive cells appeared in the interstitium of the kidney
in the MRPC, MRPC/EPO and MRPC/suramin groups. (Magnification 200×). EPO, erythropoietin;
I/R, ischemia/reperfusion; MRPC, mouse renal progenital cells.

Figure 4.Expression of GFP, CD34, and E-cadherin in consecutive sections of the kidney at day
4 after I/R injury. The positive area in MRPC, MRPC/EPO and MRPC/suramin group is widely distributed.
(Magnification 200×). EPO, erythropoietin; I/R, ischemia/reperfusion; MRPC, mouse
renal progenital cells.

Figure 5.Expression of GFP, CD34, and E-cadherin in consecutive sections of the kidney at day
7 after I/R injury. The positive area in MRPC/EPO, MRPC/suramin group MRPC/EPO decreased, while the positive
area in MRPC group was still widely distributed. (Magnification 200×). EPO, erythropoietin;
I/R, ischemia/reperfusion; MRPC, mouse renal progenital cells.

Discussion

Ischemic reperfusion injury is one of the main causes of AKI and more attention has
been focused on stem cell therapy for ameliorating this injury. There has been mounting
evidence for the existence of stem cells in the adult kidney, including the glomerulus
[22], interstitium [21,24-26], tubules [8,27], and papilla [28]. In this paper we demonstrated protective roles of MRPC, MRPC/EPO and MRPC/suramin
after injection in I/R AKI C57BL/6 mice. MRPC, spindle-shaped with a large nucleus,
were purified from the kidneys of adult C57BL/6-gfp mice (see Additional file 1 and Additional file 2: Figure S2). They exhibited features of renal progenitor cells with expression of
renal progenitor markers Oct-4 and Pax-2, Wnt-4 and WT-1, which are expressed in the
renal progenitors of metanephric mesenchyme during embryonic development [29]. MRPC possessed the mesenchymal markers vimentin and α-SMA but not the epithelial
marker E-cadherin. Furthermore, there was no expression of hematogenous or endothelial
progenitor cell markers in MRPC, such as CD45 or CD34, which negated the possibility
that MRPC originated from extrarenal tissues. In addition, MRPC were multipotent for
their differentiation into osteoblast and adipocyte lineages in vitro and in vivo (see Additional file 1 and Additional file 3: Figure S3). Moreover, we studied the roles of MRPC alone and in combination with
EPO or suramin in the I/R AKI mice model. In agreement with previous studies that
showed that MKPC accelerate renal regeneration and prolong survival after ischemic
injury [15,21], these findings identify a suitable cell population, MRPC, for possible use in future
studies of cell therapy for AKI. Here, we found that the effect of MRPC/EPO or MRPC/suramin
was considerably stronger than MRPC alone very early (day 2) after injection. However,
MRPC alone played a sustaining renal regeneration role in I/R AKI C57BL/6 mice. The
reasons for this difference still remain to be clarified. A possible explanation is
MRPC/EPO or MRPC/suramin formed more CD34+ and E-cadherin+ cells with fast incorporation into renal tubules and capillaries than MRPC alone,
consistent with differentiation mechanisms that some MKPC formed vessels with red
blood cells inside (CD34+ cells) and some incorporated into renal tubules (E-cadherin+ cells) [15].

However, MRPC alone played a sustaining renal regeneration role in I/R AKI C57BL/6
mice. The reasons for this still remain to be clarified. It is interesting that whether
MRPC homed to the injured region. Our results showed that, seven days after ischemic
injury and MRPC injection, GFP fluorescence was detected in some tubules of the kidney
by immunofluorescence. One possible explanation may be based on the damaged vascular
system in I/R AKI C57BL/6 mice. Acute ischemic injury of the kidney induced hypoxia
in the injured region and, therefore, upregulated the expression of SDF-1 which attracted
CXCR4+ cells (MRPC) to mobilize to the injured region [30]. As the renal protection effect of MRPC was fast and immediate, there may be many
mechanisms involved in the recovery process. Reduction of the inflammatory response
was considered as a possible mechanism in the treatment of AKI. It was found that
MRPC reduced the post-ischemic inflammatory response and obviously decreased macrophage
infiltration, especially when combined with EPO or suramin (see Additional file 1 and Additional file 4: Figure S4).

Additional file 4: Figure S4. Inflammatory cell infiltration. Immunofluorescence of macrophage infiltration stained
with anti-F4/80 antibody (red) one, two and three days after ischemia-reperfusion
injury in the kidney treated with PBS (postive control), with MRPC, with MRPC/EPO,
or with MRPC/suramin. Nuclears are stained with DAPI (blue) (Magnification 400×).

How MRPC combine with EPO or suramin in the treatment of AKI is still not fully understood.
As we know, EPO, a glycoprotein hormone, can stimulate the formation and differentiation
of erythroid precursor cells in the bone marrow. However, further studies have been
done on the undiscovered roles of EPO on other cell types that express EPO receptors
[31-33]. Recent studies have shown that there are EPO receptors on the surfaces of tubular
epithelial cells [31,34]. Furthermore, EPO plays an important role in these cells to protect kidneys against
acute injury in animal studies [31-33,35]. Mechanisms involved in this protection appear to be associated with anti-apoptotic,
anti-oxidative and anti-inflammatory properties as well as with the proangiogenic
potential of EPO [31]. It was reported that rhEPO treatment significantly attenuated the upregulation of
transforming growth factor 1 (TGF-1) and α-SMA and the downregulation of E-cadherin
in the obstructed kidney in a mouse model [36]. Further, EPO treatment can increase the expression of CD34 [37] after adriamycin-induced kidney injury. Moreover, E-cadherin is highly positively
regulated by EPO in a PI3K-dependent manner in CD34+ progenitor cells [38]. These findings may explain the greater improvement in renal histology and function
in the mice treated with MRPC/EPO than in those treated with MRPC alone very early
after injection. Suramin, a common drug in the treatment of trypanosomiasis, has recently
been found to be useful in accelerating kidney recovery after AKI although the exact
mechanism is still incompletely known. Recently, it was reported that the death of
renal epithelial cells could directly cause necrosis of renal fibroblasts by releasing
ATP immediately into the interstitium of the kidney as a death factor and the P2X7 receptor as a crucial mediator [39]. Since peritubular fibroblasts in the kidney are the major EPO-producing cells, inhibition
of P2X7 may promote renal structural and functional recovery after AKI. Since suramin is
a general P2 inhibitor, it can inhibit the P2X7 receptor to prevent the death of renal fibroblasts and then raise the EPO level during
the AKI process. Thus, suramin may protect against kidney injury by increasing EPO
production. There is a close intrinsic correlation between EPO and suramin. However,
it is still unclear how MRPC combine with EPO or suramin in the treatment of AKI and
advanced research work needs to be done.

Recently, some studies have proven that the therapeutic efficiency of MSC in AKI and
many other diseases may be improved by combination with a molecular treatment. La
Manna et al. [40] showed that hyaluronan monoesters with butyric acid (HB) act as a preconditioning
agent increasing angiogenesis and vascular regeneration efficiency of FMhMSCs. Mias
et al. [41] found that pretreatment with melatonin could increase the survival, paracrine activity
and efficiency of MSCs. Similarly, the protective effects of EPO compounds and MSC
combinations are supported by a study which evaluated the effect of this combination
on a rat model of ischemia [42]. Although these data are from MSC, it is still reasonable to speculate that the efficiency
of MRPC may also be enhanced by combination with molecular treatment. Our data show
that MRPC treatment was an efficient approach for recovery from injury. There was
no teratoma formed in the kidney six weeks after MRPC injection (see Additional file
1), and there are currently no reports about tumor genesis originating from MRPC. Moreover,
our data show that combined MRPC/EPO and MRPC/suramin treatment was a more efficient
approach for recovery from injury than MRPC alone very early (day 2) after injection
and that MRPC alone played a sustaining renal repair role in I/R AKI C57BL/6 mice.
Even though this potentiated effect might be related to the addition of independent
beneficial effects of the treatment agents, combination of stem cell-based therapy
with pharmacy therapy might offer a novel therapeutic approach for the treatment of
I/R-induced AKI in humans.

Conclusions

Taken together, our data suggest that MRPC, generated from the kidney of C57BL/6-gfp
mice, might provide a new approach for the treatment of AKI in an in vivo model of acute kidney injury. Our results also indicate that MRPC/EPO or MRPC/suramin
provided more beneficial effects very early (day 2) after injection, while MRPC alone
played a sustaining role in renal regeneration in the treatment of I/R AKI. These
findings suggest that it is feasible to rescue renal damage by the injection of MRPC
alone, MRPC/EPO or MRPC/suramin in mice.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

XH participated in manuscript writing, data analysis and interpretation, collection
and assembly of data. LZ participated in collection and assembly of data, data analysis
and interpretation. GL and JG participated in collection and assembly of data. YZ
participated in data analysis and interpretation. SZ participated in collection and
assembly of data. MY and YL participated in the provision of study material. FK participated
in administrative support. ZX participated in manuscript writing. SZ participated
in conception and design, financial support and final approval of manuscript. All
authors read and approved the final manuscript for publication.

Acknowledgments

The study was supported by grants from The National Natural Science Foundation of
China (Project approval number 30840079) and Shandong province science and technology
plan (Project approval number 2006GG2202002 and 2008GG10002015).